Our ability to effectively treat malaria is threatened by increasingly widespread resistance to the limited number of frontline antimalarial drugs available. Consequently, new strategies guiding prioritization of novel targets for drug development are critically needed. Here we propose targeting mitochondrial function as a strategy that could yield diverse solutions for treating malaria. The long-term goal is to define the mitochondrial proteome of the human malaria parasite, P. falciparum, and establish the core subset of these proteins that are essential for parasite survival. We envision that this knowledge will contribute to the development of new antimalarial drugs with novel mechanisms of action, and that circumvent existing modes of drug resistance. The objectives of the present research are to: (1) develop a pipeline for prioritizing then validating the set of nuclear- encoded protein that are trafficked to the mitochondrion; and (2) classify the essentiality of these proteins. The central hypothesis is that mitochondrial function is critical for malaria parasite survival during blood, mosquito and liver stages. Defining essential mitochondrial proteins and biological processes should provide new insights into fundamental parasite biology. Additionally, this can create opportunities for developing antimalarial drugs that simultaneously target blood, transmission, and liver stages of the parasite life cycle. Drugs with these characteristics are critical to malaria elimination efforts. To accomplish the objectives of this proposal, we will pursue two aims. In Specific Aim 1, we will use bioinformatics tools to predict mitochondrial targeting sequences (MTS) in nuclear-encoded proteins, and create prioritized lists of putative mitochondrial proteins. We will create libraries of MTS candidates fused to a fluorescent reporter protein and use high throughput and high content imaging to determine whether a candidate MTS is sufficient to mediate protein trafficking to the mitochondrion. In Specific Aim 2, we will use a newly developed functional genetics toolkit to epitope tag and conditionally regulate the expression level of endogenous proteins putatively trafficked to the mitochondrion. This will allow us to establish whether native proteins associated with a given MTS are indeed trafficked to the mitochondrion. Conditionally regulating the expression of these proteins will additionally facilitate assessment of which mitochondrial proteins are essential for parasite survival. Our approach is innovative because it integrates several complementary technologies-combinatorial reporter library synthesis and parasite line generation, high content imaging and functional genetics-to gain insight into mitochondrial biology on an unprecedented scale in P. falciparum. The proposed research is significant because it will provide definitive insights into the composition and vital components of this relatively enigmatic parasite organelle. Simultaneously, it will motivate targeted interference with mitochondrial function as a strategy for identifying life cycle stage-independent antimalarial drugs. These outcomes are expected to positively impact human health by improving the tools available for malaria treatment and eradication.